The mechanism of the induction of glucokinase, the enzyme that catalyzes the initial step of glycolysis and plays a major role in glucose homeostasis, will be studied. An understanding of this mechanism is a prerequisite to our understanding defects in the expression of this enzyme occurring in diabetes mellitus and neoplasm. Previously developed cloned cDNA, complimentary to the mRNA coding for rat glucokinase, will be characterized and its nucleic acid sequence determined. The cDNA, as contained in the plasmid pGK1, will be used to study the mRNA coding for glucokinase by northern blot analyses. Using pGK1, the level of mRNA coding for glucokinase will be determined in primary cultures of adult rat hepatocytes following a variety of hormonal and nutritional manipulations of the culture medium. These include an examination of the effects of insulin, dexamethasone, thyroid hormone, and glucagon; the effects of carbohydrate metabolism will be studied as well. The above manipulations will be examined alone and in combination with one another. The time course of changes in the amount of mRNA coding for glucokinase, as measured by dot-blot hybridization studies using pGK1, will be compared to the time course of changes in the activity of the enzyme, its rate of synthesis, and the level of translatable mRNA coding for the enzyme. The mechanism of the above changes in the level of the mRNA will be determined using the cloned cDNA. Specific mechanisms to be examined include transcription, turnover of the mRNA, changes in the processing of the message, examination of different pools of message within the cell, and a study of translational parameters associated with the mRNA coding for glucokinase. Our more long term aim is to study the structure of the coding and regulatory regions of the gene coding for glucokinase and experiments along these lines will be started. In particular, a full-length cDNA to the mRNA coding for glucokinase will be synthesized and used as a probe to screen a genomic library for the gene of interest. The results of this project will help define the cellular processes which act to regulate the metabolism of carbohydrates.